DNA encoding human AlkB

ABSTRACT

A human hABH polypeptide and DNA (RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptide for the treatment of mutations and the treatment of diseases which result from damaged DNA, for example, cancer. Antagonists against such polypeptides and their use as a therapeutic to augment chemotherapy of cancer cells are also disclosed.

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. More particularly, the polypeptide of the presentinvention is human homolog of the bacterial AlkB gene, sometimeshereinafter referred as "hABH". The invention also relates to inhibitingthe action of such polypeptides.

Alkylating agents induce DNA damage which may cause either killing ofcells or induction of mutation and cancer. Most of such damage issubjected to common cellular DNA repair mechanisms, such as excisionrepair and postreplication repair (Hanawalt, P. C., et al, Annu. Rev.Biochem., 48:783-836 (1979) and Witkem, Bacteriol. Rev., 40:869-907(1976)). A repair mechanism is that performed by the human DNA mismatchrepair protein.

Certain strain of E. Coli mutants have been found to be specificallysensitive to alkylating agents. Two types of such mutants have beenisolated, alkA and tagA (Yamamato, Y., et al., J. Bacteriology,135:144-152 (1978) and Karran, P., et al., J. Mol. Biol., 40:101-127(1980)). These genes control the formation of enzymes that catalyze theliberation of certain alkylated bases from damaged DNA (Karran, P.,Nature (London), 296:770-773 (1982)). In addition, ada and adc mutantshave been isolated which are defective in controlling mechanisms toinduce the adaptive response to alkylating agents (Jeggo, P., J.Bacteriol., 139:783-791 (1982)).

The tagA gene has been mapped to an E. Coli chromosome and controls aconstitutive enzyme 3-methyladenine-DNA glycosylase I that releases3-methyladenine from alkylated DNA (Karran, P. et al., Nature (London),296:770-773 (1982)). The alkA gene has also been mapped and it toocontrols an inducible enzyme, 3-methyladenine-DNA glycosylase II, whichcatalyzes the liberation of 3-methyladenine, 3-methylguanine, and7-methylguanine from the DNA (Evensen, G. and Seeberg, E., Nature(London), 296:773-775 (1982).

Another gene of E. Coli, AlkB, has also been found to controlsensitivity to methyl methane sulfonate (MMS). The AlkB gene was locatedin a region of the chromosome near ada and adc, but is not considered anallele to these genes (Sedgwick, B., J. Bacteriol., 150:984-988 (1982)).

Thus, AlkB resides in a new gene that is near the nalA gene. The AlkBphenotype is different from that of ada, since the AlkB mutant exhibiteda normal adaptive response to n-methyl-n'-nitro-n-nitrosoguanidine(Kataoka, H., et al., J. Bact., 153:1301-1307 (1983)). The AlkB gene ofE.coli has been found to be responsible for the repair of alkylated DNA(Kondo, H., et al., J. Biol. Chem., 15:1-6, (1986)).

Due to the amino acid sequence between AlkB from E.coli, the presentpolynucleotide and deduced polypeptide have been putatively identifiedas a human homolog of the E.coli AlkB protein.

In accordance with one aspect of the present invention, there isprovided a novel mature polypeptide which is hABH, as well as fragments,analogs and derivatives thereof. The polypeptide of the presentinvention is of human origin.

In accordance with another aspect of the present invention, there areprovided polynucleotides (DNA or RNA) which encode such polypeptides.

In accordance with yet a further aspect of the present invention, thereis provided a process for producing such polypeptide by recombinanttechniques.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptide, or polynucleotideencoding such polypeptide for therapeutic purposes, for example, forrepairing alkylated DNA and accordingly preventing or treating celldeath and cancer.

In accordance with yet a further aspect of the present invention, thereare provided antibodies against such polypeptides.

In accordance with yet another aspect of the present invention, thereare provided antagonists to such polypeptides, which may be used toinhibit the action of such polypeptides, for example, to prevent thispolypeptide from repairing tumor cell DNA during chemotherapy withalkylating agents.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

Collectively FIGS. 1A-1G (FIG. 1A is the first portions of thepolynucleotide and polypeptide sequences, of SEQ ID NOS: 1 and 2,respectively, FIG. 1B continues with the second sequence portions, andFIGS. 1C-1G continue in like manner to the ends of the samepolynucleotide and polypeptide sequences) show the cDNA sequence (SEQ IDNO:1) and corresponding deduced amino acid sequence (SEQ ID NO:2) forhABH. The amino acid sequence shown comprises the putative maturepolypeptide. The standard one letter abbreviations for amino acids areused in FIGS. 1A-1G to depict the polypeptide sequence.

FIG. 2 is a schematic illustration of the survival rate of cells in thepresence of increasing concentrations of MMS (methyl methane sulfonate).Cells which are wild type for AlkB show no decrease in survival rate asthere is an increase in MMS. Mutations (MT) show a dramatic decrease inthe survival rate as the concentration of MMS increases. Cells whichhave the hABH present therein show an increased survival rate ascompared to mutant cells.

FIG. 3 illustrates amino acid homology between hABH (top) and AlkB(bottom) from E.coli.

In accordance with an aspect of the present invention, there is providedan isolated nucleic acid (polynucleotide) which encodes for the maturepolypeptide having the deduced amino acid sequence of FIG. 1 (SEQ IDNO:2) or for the mature polypeptide encoded by the cDNA of the clonedeposited as ATCC Deposit No. 75855 on Aug. 9, 1994.

A polynucleotide encoding a polypeptide of the present invention may beobtained from a human prostate, testis, placenta and heart. Thepolynucleotide of this invention was discovered in a cDNA libraryderived from a human synovial sarcoma. It is structurally related to E.Coli AlkB. It contains an open reading frame encoding a protein of 307amino acid residues. The protein exhibits the highest degree of homologyto E.coli AlkB with 23% identity and 52% similarity over a 283 aminoacid stretch.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in FIG. 1 (SEQ ID NO:1) or that of thedeposited clone or may be a different coding sequence which codingsequence, as a result of the redundancy or degeneracy of the geneticcode, encodes the same mature polypeptide as the DNA of FIG. 1 (SEQ IDNO:1) or the deposited cDNA.

The polynucleotide which encodes for the mature polypeptide of FIG. 1(SEQ ID NO:2) or for the mature polypeptide encoded by the depositedcDNA may include: only the coding sequence for the mature polypeptide;the coding sequence for the mature polypeptide and additional codingsequence such as a leader or secretory sequence or a proproteinsequence; the coding sequence for the mature polypeptide (and optionallyadditional coding sequence) and non-coding sequence, such as introns ornon-coding sequence 5' and/or 3' of the coding sequence for the maturepolypeptide.

Thus, the term "polynucleotide encoding a polypeptide" encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 1 (SEQ ID NO:2) or the polypeptide encoded by the cDNA of thedeposited clone. The variant of the polynucleotide may be a naturallyoccurring allelic variant of the polynucleotide or a non-naturallyoccurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature polypeptide as shown in FIG. 1 (SEQ ID NO:2) or the same maturepolypeptide encoded by the cDNA of the deposited clone as well asvariants of such polynucleotides which variants encode for a fragment,derivative or analog of the polypeptide of FIG. 1 or the polypeptideencoded by the cDNA of the deposited clone. Such nucleotide variantsinclude deletion variants, substitution variants and addition orinsertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIG. 1 (SEQ ID NO:1) or of the coding sequence of the depositedclone. As known in the art, an allelic variant is an alternate form of apolynucleotide sequence which may have a substitution, deletion oraddition of one or more nucleotides, which does not substantially alterthe function of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the codingsequence for the mature polypeptide may be fused in the same readingframe to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5' amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains.

Thus, for example, the polynucleotide of the present invention mayencode for a mature protein, or for a protein having a prosequence orfor a protein having both a prosequence and a presequence (leadersequence).

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexahistidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The term "gene" means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

Fragments of the full length gene of the present invention may be usedas a hybridization probe for a cDNA library to isolate the full lengthcDNA and to isolate other cDNAs which have a high sequence similarity tothe gene or similar biological activity. Probes of this type preferablyhave at least 30 bases and may contain, for example, 50 or more bases.The probe may also be used to identify a cDNA clone corresponding to afull length transcript and a genomic clone or clones that contain thecomplete gene including regulatory and promotor regions, exons, andintrons. An example of a screen comprises isolating the coding region ofthe gene by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 70%,preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term"stringent conditions" means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNAs of FIG. 1 (SEQ ID NO:1) orthe deposited cDNA(s).

Alternatively, the polynucleotide may have at least 20 bases, preferably30 bases, and more preferably at least 50 bases which hybridize to apolynucleotide of the present invention and which has an identitythereto, as hereinabove described, and which may or may not retainactivity. For example, such polynucleotides may be employed as probesfor the polynucleotide of SEQ ID NO:1, for example, for recovery of thepolynucleotide or as a diagnostic probe or as a PCR primer.

Thus, the present invention is directed to polynucleotides having atleast a 70% identity, preferably at least 90% and more preferably atleast a 95% identity to a polynucleotide which encodes the polypeptideof SEQ ID NO:2 as well as fragments thereof, which fragments have atleast 30 bases and preferably at least 50 bases and to polypeptidesencoded by such polynucleotides.

The deposit(s) referred to herein will be maintained under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicro-organisms for purposes of Patent Procedure. These deposits areprovided merely as convenience to those of skill in the art and are notan admission that a deposit is required under 35 U.S.C. §112. Thesequence of the polynucleotides contained in the deposited materials, aswell as the amino acid sequence of the polypeptides encoded thereby, areincorporated herein by reference and are controlling in the event of anyconflict with any description of sequences herein. A license may berequired to make, use or sell the deposited materials, and no suchlicense is hereby granted. However, if a patent should issue which isdirected to the present invention, upon the issuance of such a patentthe deposited strain of ATCC 75855 will be irrevocably and withoutrestriction released to the public, excepting for those restrictionspermitted by enforcement of the patent.

The present invention further relates to an hABH polypeptide which hasthe deduced amino acid sequence of FIG. 1 or which has the amino acidsequence encoded by the deposited cDNA, as well as fragments, analogsand derivatives of such polypeptide.

The terms "fragment," "derivative" and "analog" when referring to thepolypeptide of FIG. 1 or that encoded by the deposited cDNA, means apolypeptide which retains essentially the same biological function oractivity as such polypeptide. Thus, an analog includes a proproteinwhich can be activated by cleavage of the proprotein portion to producean active mature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 1 or thatencoded by the deposited cDNA may be (i) one in which one or more of theamino acid residues are substituted with a conserved or non-conservedamino acid residue (preferably a conserved amino acid residue) and suchsubstituted amino acid residue may or may not be one encoded by thegenetic code, or (ii) one in which one or more of the amino acidresidues includes a substituent group, or (iii) one in which the maturepolypeptide is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol), or (iv) one in which the additional amino acids are fused tothe mature polypeptide, such as a leader or secretory sequence or asequence which is employed for purification of the mature polypeptide ora proprotein sequence. Such fragments, derivatives and analogs aredeemed to be within the scope of those skilled in the art from theteachings herein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term "isolated" means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polypeptides of the present invention include the polypeptide of SEQID NO:2 (in particular the mature polypeptide) as well as polypeptideswhich have at least 70% similarity (preferably at least 70% identity) tothe polypeptide of SEQ ID NO:2 and more preferably at least 90%similarity (more preferably at least 90% identity) to the polypeptide ofSEQ ID NO:2 and still more preferably at least 95% similarity (stillmore preferably at least 95% identity) to the polypeptide of SEQ ID NO:2and also include portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

As known in the art "similarity" between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the hABH genes. The culture conditions, suchas temperature, pH and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila and Sf9;animal cells such as CHO, COS or Bowes melanoma; plant cells, etc. Theselection of an appropriate host is deemed to be within the scope ofthose skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are PKK232-8 and PCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation. (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), thedisclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,U.S.A.). These pBR322 "backbone" sections are combined with anappropriate promoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5' flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The hABH polypeptides can be recovered and purified from recombinantcell cultures by methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

The hABH polypeptide of the present invention may be employed to protectagainst cellular DNA damage as a result of exposure to chemicalmutagens. More particularly, the hABH may be used to repair cellularDNA, such as by excision repair, substitution, removing alkylatedportion of bases or postreplication repair.

In this manner, the hABH polypeptide of the present invention may beused to treat diseases characterized by abnormal cellulardifferentiation, for example, cancer. Further, mutated DNA leads to ahost of other known and unknown disorders which could be treated withthe hABH polypeptide of the present invention.

The present invention also provides a diagnostic assay for detectingmutated hABH genes, which is indicative of a susceptibility to mutationof DNA by various agents, such as chemical mutation. One example of suchan assay is the RT-PCR method. For RT-PCR (Reverse TranscriptasePolymerase Chain Reaction), the mRNA encoding hABH is isolated from thetotal cellular RNA removed from a cell sample. The coding region of theRT-PCR products are then sequenced and compared to the hABH gene todetect mutations. Alternatively, oligonucleotide probes may be preparedwhich are highly specific for the mRNA to be detected. Sucholigonucleotide probes have between 10 and 40 base pairs and prefereblybetween 10 and 30 base pairs. The oligonucleotide probes may belabelled, for example by radioactivity. The probe is hybridized, forexample in situ hybridization, to a cDNA library prepared from totalmRNA in a cell sample derived from a host. If there is hybridization,the probe may be removed and the gene to which it hybridizes issequenced to detect mutations.

The present invention also relates to an assay which demonstrates thebiological activity of the hABH gene to protect against the effects ofexposure to chemical mutagens and alkylating agents. An example of thistype of assay comprises exposing three different groups of E. Coli cellsto varying concentrations of an alkylating agent, for example MMS. Onecell type is an HK81 strain of E. Coli which is wild-type for the alkBgene. Another cell type is an KH82 strain of E. Coli which is a mutantstrain for the alkB gene. The third group is the HK82 strain which hasbeen transfected with a vector containing the hABH gene. A survivalpercentage of these groups of E. Coli cells is then computed and theresults are shown in FIG. 2. It is clear from FIG. 2 that the mutantstrain (mt) had the lowest survival rate, while the wild-type (wt)strain had the highest survival rate. The results further show that thehABH gene was able to increase the survival rate of the mutant strainand, therefore, effectively protect against alkylating agents byrepairing DNA.

Alternatively, mammalian cells may be employed wherein cells which arewild-type and mutant for the alkB gene may be used. The mutant strainmay then be transfected with the hABH gene and percentage of survivingcells calculated. In another embodiment, knock-out mice may be employedwherein the alkB gene has been removed through genetic engineeringtechniques known to those of skill in the art.

The above-described assay could be used to identify agonist orantagonist compounds. An example of such an assay comprises preparinggroups of cells, E. Coli or mammalian, wherein one group is wild-typeand the other group is mutant for the alkB gene. The cells are thenexposed to the varying amounts of MMS as above. However, in this assaycompounds are added to the reaction and the ability of the compound toincrease or decrease the survival rate of the mutant strain could thenbe determined using an assay performed in the absence of any compoundsas a control.

Example of potential antagonists to hABH include antibodies, or in somecases an oligonucleotide, which binds to the hABH to eliminate itsfunction. Potential antagonists also include proteins closely related tohABH such that they recognize and bind to the damaged bases of the DNAbut do not repair them.

Another potential antagonist includes an antisense construct preparedusing antisense technology. Antisense technology can be used to controlgene expression through triple-helix formation or antisense DNA or RNA,both of which methods are based on binding of a polynucleotide to DNA orRNA. For example, the 5' coding portion of the polynucleotide sequence,which encodes for the mature polypeptides of the present invention, isused to design an antisense RNA oligonucleotide of from about 10 to 40base pairs in length. A DNA oligonucleotide is designed to becomplementary to a region of the gene involved in transcription (triplehelix--see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al,Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)),thereby preventing transcription and the production of hABH. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into the hABH (antisense--Okano, J.Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of hABH.

Potential antagonists also include small molecules which bind to andoccupy the effective site of the hABH polypeptide such that it isinaccessible to damaged DNA. Examples of small molecules include but arenot limited to small peptides or peptide like molecules.

The antagonists may be employed to specifically target tumor cells andprevent hABH from repairing the DNA of the tumor cell so that the resultof chemotherapy with alkylating agents is not offset. However, it isdesirable for normal cells to have the alkylated bases repaired by hABH,therefore, the above antagonists are only effective if specificallytargeted to tumor cells, or other cells which are the object ofchemotherapy. The antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as hereinafter described.

The polypeptides and agonists and antagonists may be employed incombination with a suitable pharmaceutical carrier. Such compositionscomprise a therapeutically effective amount of the polypeptide, and apharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides of the present invention may be employed in conjunctionwith other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner such as by the oral, topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes.Pharmaceutical compositions are administered in an amount which iseffective for treating and/or prophylaxis of the specific indication. Ingeneral, the pharmaceutical compositions will be administered in anamount of at least about 10 μg/kg body weight and in most cases theywill be administered in an amount not in excess of about 8 mg/Kg bodyweight per day. In most cases, the dosage is from about 10 μg/kg toabout 1 mg/kg body weight daily, taking into account the routes ofadministration, symptoms, etc.

The hABH polypeptides and agonists or antagonists may also be employedin accordance with the present invention by expression of suchpolypeptides in vivo, which is often referred to as "gene therapy."

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

The vector includes one or more promoters. Suitable promoters which maybe employed include, but are not limited to, the retroviral LTR; theSV40 promoter; and the human cytomegalovirus (CMV) promoter described inMiller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or anyother promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, pol III, andβ-actin promoters). Other viral promoters which may be employed include,but are not limited to, adenovirus promoters, thymidine kinase (TK)promoters, and B19 parvovirus promoters. The selection of a suitablepromoter will be apparent to those skilled in the art from the teachingscontained herein.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, ψ-2,ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs.5-14 (1990), which is incorporated herein by reference in its entirety.The vector may transduce the packaging cells through any means known inthe art. Such means include, but are not limited to, electroporation,the use of liposomes, and CaPO₄ precipitation. In one alternative, theretroviral plasmid vector may be encapsulated into a liposome, orcoupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the polypeptides.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

This invention is also related to the use of the gene of the presentinvention as a diagnostic. Detection of a mutated form of the gene willallow a diagnosis of a disease or a susceptibility to a disease whichresults from underexpression of hABH.

Individuals carrying mutations in the gene of the present invention maybe detected at the DNA level by a variety of techniques. Nucleic acidsfor diagnosis may be obtained from a patient's cells, including but notlimited to blood, urine, saliva, tissue biopsy and autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986))prior to analysis. RNA or cDNA may also be used for the same purpose. Asan example, PCR primers complementary to the nucleic acid encoding hABHcan be used to identify and analyze mutations. For example, deletionsand insertions can be detected by a change in size of the amplifiedproduct in comparison to the normal genotype. Point mutations can beidentified by hybridizing amplified DNA to radiolabeled RNA oralternatively, radiolabeled antisense DNA sequences. Perfectly matchedsequences can be distinguished from mismatched duplexes by RNase Adigestion or by differences in melting temperatures.

Sequence differences between the reference gene and genes havingmutations may be revealed by the direct DNA sequencing method. Inaddition, cloned DNA segments may be employed as probes to detectspecific DNA segments. The sensitivity of this method is greatlyenhanced when combined with PCR. For example, a sequencing primer isused with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., PNAS, U.S.A., 85:4397-4401(1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic assay for detectingaltered levels of the polypeptide of the present invention in varioustissues. Assays used to detect levels of the polypeptide of the presentinvention in a sample derived from a host are well-known to those ofskill in the art and include radioimmunoassays, competitive-bindingassays, Western Blot analysis and preferably an ELISA assay. An ELISAassay initially comprises preparing an antibody specific to the hABHantigen, preferably a monoclonal antibody. In addition a reporterantibody is prepared against the monoclonal antibody. To the reporterantibody is attached a detectable reagent such as radioactivity,fluorescence or in this example a horseradish peroxidase enzyme. Asample is now removed from a host and incubated on a solid support, e.g.a polystyrene dish, that binds the proteins in the sample. Any freeprotein binding sites on the dish are then covered by incubating with anon-specific protein such as bovine serum albumin. Next, the monoclonalantibody is incubated in the dish during which time the monoclonalantibodies attached to any of the polypeptide of the present inventionattached to the polystyrene dish. All unbound monoclonal antibody iswashed out with buffer. The reporter antibody linked to horseradishperoxidase is now placed in the dish resulting in binding of thereporter antibody to any monoclonal antibody bound to the polypeptide ofthe present invention. Unattached reporter antibody is then washed out.Peroxidase substrates are then added to the dish and the amount of colordeveloped in a given time period is a measurement of the amount of thepolypeptide of the present invention present in a given volume ofpatient sample when compared against a standard curve.

A competition assay may be employed wherein antibodies specific to thepolypeptide of the present invention are attached to a solid support andlabeled hABH and a sample derived from the host are passed over thesolid support and the amount of label detected attached to the solidsupport can be correlated to a quantity of the polypeptide of thepresent invention in the sample.

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the 3'untranslated region of the gene is used to rapidly select primers thatdo not span more than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with cDNA having at least 50 or60 bases. For a review of this technique, see Verma et al., HumanChromosomes: a Manual of Basic Techniques, Pergamon Press, New York(1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice may be used to express humanized antibodies to immunogenicpolypeptide products of this invention.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described.

"Plasmids" are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

"Oligonucleotides" refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5' phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

"Ligation" refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units to T4 DNA ligase ("ligase")per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described inthe method of Graham, F. and Van der Eb, A., Virology, 52:456-457(1973).

EXAMPLE 1 Bacterial Expression and Functional Complementation of hABH

The DNA sequence encoding for hABH, ATCC #75855, is initially amplifiedusing PCR oligonucleotide primers corresponding to the 5' and 3'sequences of the hABH protein. The 5' oligonucleotide primer has thesequence 5' GCGCGTCGACATGTGTCTTCTGTCAGTG (SEQ ID NO:3) contains a SalIrestriction enzyme site (underlined) followed by 18 nucleotides of hABHcoding sequence starting from the presumed N-terminal amino acid of theprotein codon. The 3' primer has the sequence 5'GCGCAAGCTTTCATCCAGATGGCAGAAACC 3' (SEQ ID NO:4) contains complementarysequences to a HindIII site (underlined) and is followed by 20nucleotides of hABH. The restriction enzyme sites correspond to therestriction enzyme sites on the bacterial expression vector pQE-9(Qiagen, Inc. 9259 Eton Avenue, Chatsworth, Calif., 91311). pQE-9encodes antibiotic resistance (Amp^(r)), a bacterial origin ofreplication (ori), an IPTG-regulatable promoter operator (P/O), aribosome binding site (RBS), a 6-His tag and restriction enzyme sites.pQE-9 was then digested with SalI and HindIII. The amplified sequenceswere ligated into pQE-9 and were inserted in frame with the sequenceencoding for the histidine tag and the RBS. The ligation mixture wasthen used to transform E. coli mutant strain HK82 by the proceduredescribed in Sambrook, J. et al., Molecular Cloning: A LaboratoryManual, Cold Spring Laboratory Press, (1989). Transformants areidentified by their ability to grow on LB plates and ampicillinresistant colonies were selected. Plasmid DNA was isolated and confirmedby restriction analysis.

The AlkB mutant strain of HK82 was then examined for its ability tocomplement an AlkB mutant. Wild-type E. Coli strain HK81 harboring thepQE-9 vector and mutant E. Coli strain HK82 containing the vectorpQE-9hABH were grown to 2×10⁸ cells per milliliter in LB ampicillinmedium at 37 degrees C. The cells were then diluted with M9 salts, andplated on LB ampicillin plates containing 0, 0,001, 0.02, and 0.03% ofMMS. The plates were incubated at 37 degree C. overnight. The resultsare depicted in FIG. 2.

EXAMPLE 2 Expression of Recombinant hABH in COS Cells

The expression of plasmid, hABH HA is derived from a vector pcDNAI/Amp(Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillinresistance gene, 3) E.coli replication origin, 4) CMV promoter followedby a polylinker region, a SV40 intron and polyadenylation site. A DNAfragment encoding the entire hABH precursor and a HA tag fused in frameto its 3' end was cloned into the polylinker region of the vector,therefore, the recombinant protein expression is directed under the CMVpromoter. The HA tag correspond to an epitope derived from the influenzahemagglutinin protein as previously described (I. Wilson, H. Niman, R.Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767).The infusion of HA tag to our target protein allows easy detection ofthe recombinant protein with an antibody that recognizes the HA epitope.

The plasmid construction strategy is described as follows:

The DNA sequence encoding for hABH, ATCC #75855, was constructed by PCRon the original EST cloned using two primers: the 5' primer 5'GCGCAAGCTTATGTGTCTTCTGTCAGTG 3' (SEQ ID NO:5) contains a HindIII site(underlined) followed by 18 nucleotides of hABH coding sequence startingfrom the initiation codon; the 3' primer sequence 5'GCGCGAATTCTCAAGCGTAGTCTGGGACGTCGTATGGGTATCCAGATGGCAGAAACC 3' (SEQ ID NO:6) contains anEcoRI site, complementary sequences to a translation stop codon, HA tagand the last 17 nucleotides of the hABH coding sequence (not includingthe stop codon). Therefore, the PCR product contains a HindIII site,hABH coding sequence followed by HA tag fused in frame, a translationtermination stop codon next to the HAtag, and an EcoRI site. The PCRamplified DNA fragment and the vector, pcDNAI/Amp, were digested withHindIII and EcoRI restriction enzyme and ligated. The ligation mixturewas transformed into E. coli strain SURE (available from StratageneCloning Systems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037)the transformed culture was plated on ampicillin media plates andresistant colonies were selected. Plasmid DNA was isolated fromtransformants and examined by restriction analysis for the presence ofthe correct fragment. For expression of the recombinant hABH, COS cellswere transfected with the expression vector by DEAE-DEXTRAN method. (J.Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A LaboratoryManual, Cold Spring Laboratory Press, (1989)). The expression of thehABH HA protein was detected by radiolabelling and immunoprecipitationmethod. (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, (1988)). Cells were labelled for 8 hourswith ³⁵ S-cysteine two days post transfection. Culture media were thencollected and cells were lysed with detergent (RIPA buffer (150 mM NaCl,1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5). (Wilson, I.et al., Id. 37:767 (1984)). Both cell lysate and culture media wereprecipitated with a HA specific monoclonal antibody. Proteinsprecipitated were analyzed on 15% SDS-PAGE gels.

EXAMPLE 3 Expression Pattern of hABH in Human Tissue

Northern blot analysis was carried out to examine the levels ofexpression of hABH in human tissues. Total cellular RNA samples wereisolated with RNAzol™ B system (Biotecx Laboratories, Inc. 6023 SouthLoop East, Houston, Tex. 77033). About 10 μg of total RNA isolated fromeach human tissue specified was separated on 1% agarose gel and blottedonto a nylon filter. (Sambrook, Fritsch, and Maniatis, MolecularCloning, Cold Spring Harbor Press, (1989)). The labeling reaction wasdone according to the Stratagene Prime-It kit with 50 ng DNA fragment.The labeled DNA was purified with a Select-G-50 column. (5 Prime--3Prime, Inc. 5603 Arapahoe Road, Boulder, Colo. 80303). The filter wasthen hybridized with radioactive labeled full length hABH gene at1,000,000 cpm/ml in 0.5M NaPO₄, pH 7.4 and 7% SDS overnight at 65° C.After wash twice at room temperature and twice at 60° C. with 0.5×SSC,0.1% SDS, the filter was then exposed at -70° C. overnight with anintensifying screen. The message RNA for hABH is abundant in thymus,testis, gall bladder, liver, prostate, heart and placenta.

EXAMPLE 4 Expression via Gene Therapy

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention is amplifiedusing PCR primers which correspond to the 5' and 3' end sequencesrespectively. The 5' primer containing an EcoRI site and the 3' primer$further includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified $EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HB101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interestproperly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellsare transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 6                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1953 BASE PAIRS                                                   (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGGAAGATGGCAGCGGCCGTGGGCTCTGTGGCGACTCTGGCGACTGAGCCCGGGGAGGAC60                GCCTTTCGGAAACTTTTCCGCTTCTACCGTCAGAGCCGGGCCCGGGACCGCAGACCTGGA120               AGGGGTCATCGACTTCTCGGCGGCCCACGCAGCCCGTGCAAGGGTCCTGGTGCCCAAAAG180               GTGATCAAATCTCAGCTAAATGTGTCTTCTGTCAGTGAGCAGAATGCATATAGAGCAGGT240               CTTCAGCCCGTCAGCAAGTGGCAAGCCTATGGACTCAAAGGCTATCCTGGGTTTATTTTT300               ATCCCAAACCCCTTCCTCCCAGGTTACCAGTGGACACTGGGTGAAACAGTGCCTTAAGTT360               ATATTCCCAGAAACCTAATGTATGTAACCTGGACACACACATGTCTAAAGAAGAGACCCA420               AGATCTGTGGGAACAGAGCAAAGAGTTCCTGAGGTATAAAGAAGCGACTAAACGGAGACC480               CCGAAGTTTACTGGAGAAACTGCGTTGGGTGACCGTAGGCTACCATTATAACTGGGACAG540               TAAGAAATACTCAGCAGATCATTACACACCTTTCCCTTCTGACCTGGGTTTCCTCTCAGA600               GCAAGTAGCCGCTGCCTGTGGATTTGAGGATTTCCGAGCTGAAGCAGGGATCCTGAATTA660               CTACCGCCTGGACTCCACACTGGGAATCCACGTAGACAGATCTGAGCTAGATCACTCCAA720               ACCCTTGCTGTCATTCAGCTTTGGACAGTCCGCCATCTTTCTCCTGGGTGGTCTTCAAAG780               GGATGAGGCCCCCCCGCCCATGTTTATGCACAGTGGTGACATCATGATAATGTCGGGTTT840               CAGCCGCCTCTTGAACCACGCAGTCCCTCGTGTCCTTCCAAATCCAGAAGGGGAAGGCCT900               GCCTCACTGCCTAGAGGCACCTCTCCCTGCTGTCCTCCCGAGAGATTCAATGGTAGAGCC960               TTGTTCTATGGAGGACTGGCAGGTGTGTGCCAGCTACTTGAAGACCGCTCGTGTTAACAT1020              GACTGTCCGACAGGTCCTGGCCACAGACCAGAATTTCCCTCTAGAACCCATCGAGGATGA1080              AAAAAAGAGACATCAGTACAGAAGGTTTCTGCCATCTGGATGACCAGAATAGCGAAGTAA1140              AACGGGCCAGGATAAACCCTGACAGCTGAGACTTGGAGATCCCATCCTTTTTACTCAGGC1200              ACCTGCTTACCGTAAATGATCATGTTATTGTGTATTGCCGTGGACTTCAGCACCCAGACA1260              AGCCAAAAACAGAGACAGGGAAGAACTCATTGTTGATCACACTGTTGCCTTGGAACCCAC1320              GCAGAAGTAAACTCATCCACTTTGCTCAGAGAAGTGTTTGACATGGTCTGTTCCTAGTTA1380              CATGTTGGCTGTAATGTATGTTGAGAAGTCAGTCCAAGGAGGTATGTTCTTCCACAACAG1440              CCTTCTCAGCCTCTGCTATTTCCTTTGAGGAAGGTAGAAGTGAGTTTCCATGTTTGCAGA1500              GTATTTAAATACCTCAGATTTTATTAATGAGAAATACAGTACCCCTCCCTCCACTCCATC1560              TGGTAATTTATGGTAAAATTGTGGTTCTGTGAACCAGCTATTAGTCTCATCTTCTTAACT1620              CCCTCAGGCATCATCAAATTCTTTGATCTTCTCTTCCACCTCTCTGGCTCTCATGGAAGA1680              ATCCTTTACACATGAAAACAATGGAACTGGAAAATCTTGTCTTTTAGAAAAGAAATTAAT1740              CACAACTATCTCTCTTGCCTAAAAGATAAATATAGGTAAACCCAAGGAAAGGGGAATTTA1800              GTTTCTCTACATGTCATTTCGGTCTCCAAACTCCCTGTTGGCTTTTTAATGCAATTTTAA1860              TTGTTGGAATAAAAAAGTCCCAAGGGTGTTTTGTTACTGTTTTCTCCATGAATAAACTCA1920              CTTGATTTTAAAAAAAAAAAAAAAAAAAAAAAA1953                                         (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 307 AMINO ACIDS                                                   (B) TYPE: AMINO ACID                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetCysLeuLeuSerValSerArgMetHisIleGluGlnValPhe                                 51015                                                                         SerProSerAlaSerGlyLysProMetAspSerLysAlaIleLeu                                 202530                                                                        GlyLeuPheLeuSerGlnThrProSerSerGlnValThrSerGly                                 354045                                                                        HisTrpValLysGlnCysLeuLysLeuTyrSerGlnLysProAsn                                 505560                                                                        ValCysAsnLeuAspThrHisMetSerLysGluGluThrGlnAsp                                 657075                                                                        LeuTrpGluGlnSerLysGluPheLeuArgTyrLysGluAlaThr                                 808590                                                                        LysArgArgProArgSerLeuLeuGluLysLeuArgTrpValThr                                 95100105                                                                      ValGlyTyrHisTyrAsnTrpAspSerLysLysTyrSerAlaAsp                                 110115120                                                                     HisTyrThrProPheProSerAspLeuGlyPheLeuSerGluGln                                 125130135                                                                     ValAlaAlaAlaCysGlyPheGluAspPheArgAlaGluAlaGly                                 140145150                                                                     IleLeuAsnTyrTyrArgLeuAspSerThrLeuGlyIleHisVal                                 155160165                                                                     AspArgSerGluLeuAspHisSerLysProLeuLeuSerPheSer                                 170175180                                                                     PheGlyGlnSerAlaIlePheLeuLeuGlyGlyLeuGlnArgAsp                                 185190195                                                                     GluAlaProProProMetPheMetHisSerGlyAspIleMetIle                                 200205210                                                                     MetSerGlyPheSerArgLeuLeuAsnHisAlaValProArgVal                                 215220225                                                                     LeuProAsnProGluGlyGluGlyLeuProHisCysLeuGluAla                                 230235240                                                                     ProLeuProAlaValLeuProArgAspSerMetValGluProCys                                 245250255                                                                     SerMetGluAspTrpGlnValCysAlaSerTyrLeuLysThrAla                                 260265270                                                                     ArgValAsnMetThrValArgGlnValLeuAlaThrAspGlnAsn                                 275280285                                                                     PheProLeuGluProIleGluAspGluLysLysArgHisGlnTyr                                 290295300                                                                     ArgArgPheLeuProSerGly                                                         305                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GCGCGTCGACATGTGTCTTCTGTCAGTG28                                                (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GCGCAAGCTTTCATCCAGATGGCAGAAACC30                                              (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GCGCAAGCTTATGTGTCTTCTGTCAGTG28                                                (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GCGCGAATTCTCAAGCGTAGTCTGGGACGTCGTATGGGTATCCAGATGGCAGAAACC57                   __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide comprising apolynucleotide having at least 95% identity to a member selected fromthe group consisting of:(a) a polynucleotide encoding a polypeptidecomprising amino acid 2 to 307 of SEQ ID NO:2; (b) the complement of(a).
 2. The isolated polynucleotide of claim 1 wherein said member is(a).
 3. The isolated polynucleotide of claim 1 wherein saidpolynucleotide encodes a polypeptide comprising amino acid 1 to 307 ofSEQ ID NO:2.
 4. The isolated polynucleotide of claim 1 comprisingnucleotides 200 to 1953 of SEQ ID NO:1.
 5. The isolated polynucleotideof claim 1 comprising a polynucleotide sequence identical to thepolynucleotide sequence of SEQ ID N0:1.
 6. The isolated polynucleotideof claim 1, wherein the polynucleotide is DNA.
 7. The isolatedpolynucleotide of claim 1, wherein said polynucleotide is RNA.
 8. Amethod of making a recombinant vector comprising inserting the isolatedpolynucleotide of claim 2 into a vector, wherein said polynucleotide isDNA.
 9. A recombinant vector comprising the polynucleotide of claim 2,wherein said polynucleotide is DNA.
 10. A recombinant host cellcomprising the polynucleotide of claim 2, wherein said polynucleotide isDNA.
 11. A method for producing a polypeptide comprising expressing fromthe recombinant cell of claim 10 the polypeptide encoded by saidpolynucleotide.
 12. An isolated polynucleotide comprising apolynucleotide having at least a 95% identity to a member selected fromthe group consisting of:(a) a polynucleotide encoding the same maturepolypeptide encoded by the human cDNA in ATCC Deposit No. 75855, and (b)the complement of (a).
 13. The isolated polynucleotide of claim 12,wherein the member is (a).
 14. The isolated polynucleotide of claim 12,wherein the member is (b).
 15. The isolated polynucleotide of claim 12comprising a polynucleotide which encodes for the same maturepolypeptide encoded by the human cDNA in ATCC Deposit No.
 75855. 16. Theisolated polynucleotide of claim 12 wherein said polynucleotidecomprises DNA identical to the coding portion of the human cDNA in ATCCDeposit No. 75855 which encodes a mature polypeptide.